<h2 class="jove_title"><a href="https://www.jove.com/video/52008">Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System</a></h2>
Kristina M. Obom1, Patrick J. Cummings1, Janelle A. Ciafardoni1, Yasunori Hashimura2, Daniel Giroux2
1Center for Biotechnology Education, Johns Hopkins University, 2PBS Biotech, Inc.
Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells. 
<h2 class="jove_title"><a href="https://www.jove.com/video/52084">Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy</a></h2>
Sabriya Stukes, Arturo Casadevall
Department of Microbiology and Immunology, Albert Einstein College of Medicine
We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.
<h2 class="jove_title"><a href="https://www.jove.com/video/51714">Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo</a></h2>
Juha T. Huiskonen, Marie-Laure Parsy, Sai Li, David Bitto, Max Renner, Thomas A. Bowden
Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford
An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.
<h2 class="jove_title"><a href="https://www.jove.com/video/52148"> Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun</a></h2>
Jason Arsenault1,2, Andras Nagy1, Jeffrey T. Henderson1, John A. O'Brien2
1Leslie Dan Faculty of Pharmacy, University of Toronto, 2MRC-Laboratory of Molecular Biology, Cambridge, UK
Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

october 2014: this month in jove - visualizing infectious disease, transfecting with the gene gun, and a novel bioreactor system

Here's a look at what's coming up in the <a href="http://www.jove.com/archive/92">October 2014 issue</a> of <a href="http://www.jove.com/">JoVE: The Journal of Visualized Experiments</a>.
Right now, the world is witnessing the worst Ebola virus outbreak in history. So this month in <a href="http://www.jove.com/i2">JoVE Immunology &#38; Infection</a>, we highlight a technique than can help us better understand how viruses establish infection. Ebola and many other enveloped viruses, including HIV, use glycoprotein spikes on their surfaces to penetrate host cells. <a href="http://www.jove.com/video/51714">Huiskonen et al.</a> use a computational approach to analyze viral envelope spikes, revealing their precise, 3D structure, which is critical for understanding the molecular interactions between certain viruses and their hosts. And this knowledge can guide the design of antiviral drugs and vaccines. Our authors demonstrate the technique with a virus from the family Bunyaviridae, but it can be applied to many other viruses that pose biological threats.
Viruses aren't the only pathogens we worry about in <a href="http://www.jove.com/i2">JoVE Immunology &#38; Infection</a>. Bacteria and fungi can be just as contagious and deadly. Every year the fungus Cryptococcus neoformans infects more than 1 million people worldwide and causes over 600,000 deaths mostly in sub-Saharan Africa, where it is one of the leading causes of death in people with AIDS. Cryptococcal cells are generally killed by macrophage cells of the immune system; but some can survive latently in macrophages, then later release into the surroundings. This phenomenon is poorly understood, so <a href="http://www.jove.com/video/52084">Stukes and Casadevall</a> have developed a way to infect macrophages with Cryptococcus in vitro and observe them over an extended period using time-lapsed microscopy. This is a promising technique for studying the complex interactions between Cryptococcus and host macrophages, and can be applied to other fungal pathogens as well.
In <a href="http://www.jove.com/neuro">JoVE Neuroscience</a>, we feature a technique that combines ballistics, or the science of shooting, with genetic engineering. Bioballistic gene guns were originally developed to inject genetic information into plant cells. <a href="http://www.jove.com/video/52148">Arsenault et al.</a> have applied this technique to mammalian tissues. Where terminally differentiated cells, like neurons, can difficult to transfect using conventional methods. By using an improved and patented bioballistic delivery method, our authors quickly and efficiently transfect various genes into distinct tissue regions.
A common goal in <a href="http://www.jove.com/bioengineering">JoVE Bioengineering</a> is to culture bacteria or eukaryotic cells in large scales for industrial or biomedical applications. Some cultures, like bacteria and yeasts, can be grown in suspension, But many mammalian cells grow in aggregates, while others grow anchored to a substrate <a href="http://www.jove.com/video/52008">Obom et al.</a> present a novel bioreactor system that can support all three types of cultures. They focus on anchorage-dependent cells, which have culture parameters that can be difficult to control, especially in large scales. Small beads called microcarriers, are added to the culture, to increase the surface area for cell growth, allowing anchorage-dependent cells to be grown in suspension. This system has tremendous potential for growing a variety of cell types and products for many applications.
You've just had a sneak peek of the <a href="http://www.jove.com/archive/92">October 2014 issue</a> of JoVE. Visit the website to see the full-length articles, plus many more, in <a href="http://www.jove.com/">JoVE: The Journal of Visualized Experiments</a>.

Watch this Scientific Journal Video about October 2014: This Month in JoVE - Visualizing Infectious Disease, Transfecting with the Gene Gun, and a Novel Bioreactor System at JoVE.com

October 2014: This Month in JoVE - Visualizing Infectious Disease, Transfecting with the Gene Gun, and a Novel Bioreactor System

Here's a look at what's coming up in the <a href="http://www.jove.com/archive/92">October 2014</a> issue of JoVE.

Here's a look at what's coming up in the October 2014 issue of JoVE: The Journal of Visualized Experiments.
Right now, the world is witnessing the worst ...

october-2014-this-month-jove-visualizing-infectious-disease

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1.9K ビュー.  Massachusetts Eye and Ear. Here's a look at what's coming up in the October 2014 issue of JoVE.

ビデオ: October 2014: This Month in JoVE - Visualizing Infectious Disease, Transfecting with the Gene Gun, and a Novel Bioreactor System

transfecting raw264.7 cells with a luciferase reporter gene

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

transient gene expression in tobacco using gibson assembly and the gene gun

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene...

regioselective biolistic targeting in organotypic brain slices using a modified gene gun

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

construction and characterization of a novel vocal fold bioreactor

Construction and Characterization of a Novel Vocal Fold Bioreactor

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem...

modeling charcot-marie-tooth disease in vitro by transfecting mouse primary motoneurons

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

preparation of gene gun bullets and biolistic transfection of neurons in slice culture

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

biolistic-mediated gene transfer: a technique to deliver gene of interest in target cells via a biolistic gene gun

In this video, we demonstrate the protocol of biolistic gene gun-mediated gene delivery in cultured cells. This method provides a simple method of stable genetic transformation using a minimal quantity of...

Biolistic-Mediated Gene Transfer: A Technique to Deliver Gene of Interest in Target Cells via a Biolistic Gene Gun

transfecting human neural stem cells with the amaxa nucleofector

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo.  This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by...

gene-gun transfection of hippocampal neurons

Gene-gun Transfection of Hippocampal Neurons

cultivation of mammalian cells using a single-use pneumatic bioreactor system

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

zika virus infectious cell culture system and the in vitro prophylactic effect of interferons

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Next, sequentially add 2 micrograms of DNA, 10 microliters of calcium chloride, and two microliters of spermidine free base into each tube of beads. Vortex each sample then, incubate the tubes at ambient temperature for 5 minutes, with occasional gentle flicking to resuspend the settled...

constructing and integrating the bioreactor and the liquid-liquid extraction system

Constructing and Integrating the Bioreactor and the Liquid-Liquid Extraction System

transfecting primary macrophages with modified mrna

This video demonstrates the process of transfecting primary macrophages with modified mRNA encoding green fluorescent protein. These modified mRNAs, designed to reduce immunogenicity and improve its stability, ensure the successful transfection and subsequent expression of green fluorescent proteins, which is confirmed through fluorescence...

Transfecting Primary Macrophages with Modified mRNA

a novel bioreactor for high density cultivation of diverse microbial communities

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal...

visualizing the calcitonin gene-related peptide immunoreactive innervation of the rat cranial dura mater with immunofluorescence and neural tracing

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

Here we present a protocol to visualize spatial correlation of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and blood vessels in the cranial dura mater using immunofluorescence and fluorescent histochemistry with CGRP and phalloidin, respectively. In addition, the origin of these nerve fibers was retrograde traced with a fluorescent neural...

visualizing efficacy of pesticides against disease vector mosquitoes in the field

Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

The efficacy of public health pesticides targeting nuisance and disease-vector insects is not uniform across different ecological zones. Here we present a system of techniques using captive vector insects as sentinels for pesticide efficacy to derive electronic maps supporting the standard evaluation of pesticides across multiple...

biocontained carcass composting for control of infectious disease outbreak in livestock

Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

Using readily available materials, this biocontained composting system enables effective on-site disposal of large animal carcasses arising in the event of infectious disease outbreak. This procedure kills most infectious agents in carcasses and contaminated manure.  Once infectious agent is confirmed non-viable, mature compost can be spread as...

recombinant collagen i peptide microcarriers for cell expansion and their potential use as cell delivery system in a bioreactor model

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor...

bimolecular fluorescence complementation (bifc) assay for protein-protein interaction in onion cells using the helios gene gun

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

August 2012: This Month in JoVE

Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and mirrors to refract and direct light to optical sensors. The August 2012 issue of JoVE (Journal of Visualized Experiments) is marked by the third publication from the Ozcan Lab (University of California, Los Angeles) on their lens free lens-free "on-chip" microscopy platform, which they have pioneered.
Back in 2009, the Ozcan lab published their first article, which demonstrated the use of a lens-free imaging platform that could image cells within whole blood. This imaging technique involves placing a sample directly on an optoelectric sensor array, like the CCD or CMOS chips found in the standard camcorder. Then, LED illumination from above the sample casts shadows on the chip, which are recorded as digital holograms that can be subjected to image processing. Not only does this lensless imaging system allow for an extremely large field of view (FOV of 0.6-8 cm2), the lack of bulky optical components makes it excellent for microfluidic applications. The Ozcan group demonstrates this in their follow-up 2011 JoVE article, in which fluorescently labeled white blood cells are imaged within a microfluidic chip.
This August in JoVE, the Ozcan group demonstrates further advancements to their lens-free microscopy platform by applying it to tomographic imaging of the nematode, C. elegans. By changing the angle of illumination, using a motorized rotation stage, the Ozcan group shows how multiple holographic slices may be generated for a sample-which, following processing, can be combined into tomograms. The Ozcan group confirms that their 3D reconstruction of the worm is successful by citing that the pharyngeal tube, a 5-micron-thick structure, is only visible in a single slice and in a 3D reconstructed cross section.
The compact and inexpensive nature of the lens-free imaging systems - designed and assembled by the Ozcan group - make them well suited for point-of-care diagnostic applications, or even cell phone based microscopy that can be performed in the field.
At the Ernest Gallo Clinic and Research Center at University of California, San Francisco JoVE learns how to visualize morphological changes in single cells using three-dimensional fluorescence microscopy and computer analysis. In this procedure, cells are given agonist, antagonist plus agonist, or no treatment, and prepared for immunofluorescence microscopy. Three-dimensional images are acquired with the help of a two-photon laser system, and the fluorescence data are processed on the computer using Imaris and Matlab.
To demonstrate the power of this technique, our authors quantify single-cell phenotypic changes in transfected HEK293 cells. Three-dimensional morphometric analysis shows dramatic changes in cells treated with agonist. These changes are not always apparent when using conventional 2D analysis. This technique allows morphological changes in single cells to be quantified and characterized in response to pharmacologic stimuli, offering a powerful new assessment tool to the arsenal of drug discovery research.
This month, in JoVE Applied Physics, we travel to South Carolina to visit the School of Materials Science and Engineering at Clemson University. Here, researchers show us how to construct lithium batteries for laboratory research.
The basic components of a lithium coin cell are the working electrode, the lithium electrolyte, and a lithium foil counter electrode. The different components of the coin cell are carefully assembled and sealed with a crimping machine. The finished coin cell can then be characterized in a battery tester. Using their homemade coin cells, our researchers can experiment with using different materials to make the cathodes of lithium coin cells.
This month in JoVE Immunology and Infection, we head to Basel, Switzerland to witness two techniques for diagnosing helminth infections. We also see how investigators perform the shuttle run test in children, in order to assess the effects of these infections on physical fitness.
Helminths are parasitic worms that can live inside humans and animals. Many species infect the intestinal tract, and may be transmitted through soil. Our authors demonstrate the Kato-Katz technique, which is used for diagnosing several common soil-transmitted helminths. They also demonstrate the Baermann technique, which uses light to stimulate the migration of certain types of phototactic helminths out of sample preparations. The various types of helminth species can subsequently be determined based on morphology.
Our authors then demonstrate how to set up and perform a 20-meter shuttle run test for children, and to measure the impact of helminth infections on physical fitness. This method can help determine the true burden of soil-transmitted helminth infections on overall health.
This brief summary highlights but a few notable video-articles that will be released in August. We'll also feature articles that demonstrate how to express genes in the midbrain of the chick embryo, study neutrophil transmigration in parallel flow chambers lined with endothelial cells, and precisely measure proteolytic degradation of the extracellular matrix.

Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and mirrors to refract and direct light to optical sensors. The August 2012 issue of JoVE (Journal of Visualized Experiments) is marked by the third publication from the Ozcan Lab (University of California, Los Angeles) on their lens-free "on-chip" microscopy platform, which they have pioneered.

Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and ...